E-Selectin (also known as Endothelial Leukocyte Adhesion Molecule-1, ELAM-1, or CD62E) is a 115 kDa, type I transmembrane glycoprotein expressed only on endothelial cells and only after activation by inflammatory cytokines (IL-1β or TNF-α) or endotoxins. Expression is transitory, reaching a maximum within about 6 hours of stimulation and then declining with shedding of soluble E-Selectin. Cell-surface E-Selectin is a mediator of the rolling attachment of leukocytes to the endothelium, an essential step in extravasation of leukocytes at the site of inflammation thereby playing a key role in localized inflammatory response. E-Selectin is believed to be particularly important in inflammation involving the skin.

The extracellular part of E-Selectin includes a calcium-dependent C2-type lectin domain, an epidermal growth factor (EGF) domain, and six repeats of a complement-regulatory-protein-like sequence. E-Selectin binds sialyl Lewis X (sLex), a sialic acid-galactose-N-acetylglucosamine-fructose tetrasaccharide, but the actual recognition is thought to be for a specific presentation of those glycosyl units in a precise three-dimensional configuration on a specific glycoprotein rather than for that particular carbohydrate.

E-Selectin is found in the blood of healthy individuals, probably arising from proteolytic cleavage of the surface-expressed molecule. Elevated levels of E-Selectin in serum have been reported in a variety of pathological conditions. Although it might be anticipated that E-Selectin would suppress leukocyte migration by competing with surface-associated E-Selectin, it may actually activate neutrophils and act as a pro-inflammatory agent.

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for E-Selectin/CD62E has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any E-Selectin/CD62E present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for E-Selectin/CD62E is added to the wells and binds to the combination of capture antibody-E-Selectin/CD62E in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of E-Selectin/CD62E present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven E-Selectin/CD62E standard dilutions and E-Selectin/CD62E sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to human E-Selectin/CD62E (8 x 12)

2. 2 vials human E-Selectin/CD62E Standard lyophilized, 6000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human E-Selectin/CD62E antibody

4. 2 vials Streptavidin-HRP solution

5. 4 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

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